Cation-mediated energy transfer in G-quadruplexes revealed by an internal fluorescent probe

Article abstract of DOI:10.1021/ja1079578

A single pyridine unit incorporated into G-quadruplex DNA has revealed efficient energy transfer reactions in cation-containing G-quadruplexes. 8-(2-Pyridyl)-2′-deoxyguanosine, "2PyG", is a highly sensitive internal fluorescent probe of G-quadruplex folding and energy transfer. 2PyG was minimally disruptive to G-quadruplex folding and exhibited intense fluorescence, even when it was base-stacked with other guanine residues. Using 2PyG we have quantified energy transfer efficiencies within G-quadruplex structures prepared under conditions of excess Na⁺/K⁺ (110 mM) or in 40% polyethylene glycol (PEG) under salt deficient conditions. G-quadruplex structures containing coordinated cations exhibited efficient DNA-to-probe energy transfer reactions (η_t = 0.11-0.41), while PEG-folded G-quadruplexes exhibited very little energy transfer (η_t = 0.02-0.07). Experiments conducted using unmodified G-quadruplexes suggest that cation coordination at the O⁶ position of guanine residues results in enhanced quantum yields of G-quadruplex nucleobases that, in turn, serve as efficient energy donors to 2PyG. Given the growing interest in G-quadruplex-based devices and materials, these results will provide important design principles toward harnessing the potentially useful photophysical properties of G-quadruplex wires and other G-rich structures.

Full text of DOI:10.1021/ja1079578

Published on Web 12/02/2010
Cation-Mediated Energy Transfer in G-Quadruplexes Revealed by an Internal
Fluorescent Probe
Anae¨lle Dumas and Nathan W. Luedtke*
Institute of Organic Chemistry, UniVersity of Zu¨rich, Winterthurerstrasse 190, CH-8057, Switzerland
Received September 3, 2010; E-mail: luedtke@oci.uzh.ch
Scheme 1. Structure and Synthesis of 8-(2-pyridyl)-2′-
Abstract: A single pyridine unit incorporated into G-quadruplex
DNA has revealed efficient energy transfer reactions in cation-
containing G-quadruplexes. 8-(2-Pyridyl)-2′-deoxyguanosine,
“2PyG”, is a highly sensitive internal fluorescent probe of G-
quadruplex folding and energy transfer. 2PyG was minimally
disruptive to G-quadruplex folding and exhibited intense fluores-
cence, even when it was base-stacked with other guanine
residues. Using 2PyG we have quantified energy transfer ef-
ficiencies within G-quadruplex structures prepared under condi-
tions of excess Na+/K+ (110 mM) or in 40% polyethylene glycol
(PEG) under salt deficient conditions. G-quadruplex structures
containing coordinated cations exhibited efficient DNA-to-probe
energy transfer reactions (ηt ) 0.11-0.41), while PEG-folded
Deoxyguanosine (2), the 2PyG Phosphoramidite (3), and a
Cation-Stabilized G-Tetrad11
G-quadruplexes exhibited very little energy transfer (ηt
)
0.02-0.07). Experiments conducted using unmodified G-quadru-
plexes suggest that cation coordination at the O6 position of
guanine residues results in enhanced quantum yields of G-
quadruplex nucleobases that, in turn, serve as efficient energy
donors to 2PyG. Given the growing interest in G-quadruplex-
based devices and materials, these results will provide important
design principles toward harnessing the potentially useful pho-
tophysical properties of G-quadruplex wires and other G-rich
structures.
energy transfer.6,7,10 To address this apparent conundrum, here we
report 8-(2-pyridyl)-2′-deoxyguanosine, “2PyG” (2), as a “turn-
on” fluorescent probe of G-quadruplex folding and energy transfer.
Using 2PyG we have quantified energy transfer efficiencies within
G-quadruplexes prepared under conditions of excess NaCl or KCl
(110 mM) or in 40% polyethylene glycol 200 (PEG) under salt-
deficient conditions (10 mM lithium cacodylate). Interestingly, the
G-quadruplex structure is necessary but not sufficient for promoting
efficient intramolecular energy transfer. The binding of ions to the
O6 positions of guanine residues is also needed for the unusually
efficient energy transfer reactions within G-quadruplex structures.
G-quadruplexes are intriguing nanostructures possessing potential
biological relevance1,2 and interesting photophysical and material
properties.3-5 Energy and electron transfer reactions involving
G-quadruplexes can mediate DNA damage and repair4 and might
facilitate the development of new molecular devices.5 Previous
studies have suggested that G-G base-stacking, hydrogen bonding,
and/or restricted motions within G-quadruplexes can enhance the
photoexcited lifetimes and energy transfer properties of guanine
residues.6 However, the poor quantum yield (Φ ≈ 10-4), short-
lived excited state (τ ≈ 1 ps), and spectral overlap of 2′-
deoxyguanosine (1) with other nucleobases prevent the quantifi-
cation of energy transfer efficiencies (ηt) within unmodified
G-quadruplexes.6
Internal fluorescent probes serving as energy acceptors can
provide powerful means to characterize energy and electron transfer
processes within DNA.7 Fluorescent base analogs, like 2-aminopu-
rine (2AP), can exhibit useful fluorescence properties when inserted
into the loops or ends of G-quadruplexes,8 but attempts by Mergny
and co-workers to introduce fluorescent guanine mimics at positions
directly involved in G-tetrad formation resulted in nonemissive
products.9 This result was consistent with numerous studies
involving duplex DNA where 2AP, 6-methylisoxanthopterin, 3-me-
thylisoxanthopterin, and other fluorescent guanine analogs were
quenched 100-fold or more by close proximity and base-stacking
interactions with purines, the same factors needed for efficient
To furnish a guanosine mimic for G-quadruplex folding and
energy transfer studies, we synthesized 8-(2-pyridyl)-2′-deoxygua-
nosine (2) (Scheme 1).11 Compound 2 exhibits a much higher
quantum yield in acetonitrile (Φ ≈ 0.71) than in water (Φ ≈ 0.02).12
The environmental sensitivity of 2 and its spectral overlap with
2′-deoxyguanosine (1) suggested that 2PyG might serve as a useful
internal fluorescent probe of G-quadruplex folding and energy
transfer.12 The corresponding 5′-DMT-3′-phosphoramidite of 2PyG
(3) was therefore synthesized in eight steps with 11% overall yield
and introduced into single sites of G-quadruplexes derived from
the human telomeric repeat “Telo” and the cKit promoter (Scheme
1).11 Importantly, 2PyG is minimally disruptive to G-quadruplex
folding and can exhibit good quantum yields (Φ ) 0.04-0.15)
even when base-stacked with other guanine residues. G-quadru-
plexes containing 2PyG can adopt wild-type folds that exhibit
efficient DNA-to-probe energy transfer reactions (ηt
)
0.11-0.41).13 Interestingly, these efficiencies are highly sensitive
to DNA folding as well as the presence of cations within the folded
G-quadruplexes.
Modification of the 8-position of guanine offers an attractive
avenue to fluorescent probes because it is not involved in the
extensive base-pairing interactions of G-tetrads (R2, Scheme 1).
Arylation of the 8-position of guanine can yield fluorescent
9
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10.1021/ja1079578  2010 American Chemical Society

C O M M U N I C A T I O N S
products,14 and 8-aryl and pyridyl derivatives have been used for
the self-assembly of intermolecular G-tetrads.3a,f,g While the
addition of bulky groups to the 8-position of guanosine can shift
the conformational equilibrium of the glycosidic bond from anti
to syn,15-17 G-quadruplex folding can force 8-modified guanosines
to adopt anti conformations with relatively small energetic penalties
to quadruplex folding (e1 kcal/mol).15b,c,e We used circular
dichroism and thermal denaturation experiments to determine the
impact of 2PyG incorporation on the global structure and stability
of G-quadruplexes carrying 2PyG. We selected sites known to be
directly involved in G-tetrad formation for modification.18,19 As
compared to the wild-type sequences, the incorporation of 2PyG
into the G9 position of Telo caused very little, if any, change to
the global secondary structure of Telo in 110 mM KCl (Figure 1A).
A very similar result was observed for cKitG10 (Figure 1B).
Consistent with the direct involvement of 2PyG in folded G-tetrads,
the substitution of G9 in Telo with 2PyG caused a significant
thermal stabilization (∆Tm ) +10 °C) of the “(3 + 1) hybrid”
structure due to the syn conformational preference of this position
(Figure 1A,G).15,17,18a In contrast, the G10 position of cKit is known
to favor an anti conformation,18b and its substitution with 2PyG
caused a small thermal destabilization (∆Tm ) -4 °C) as compared
to the unmodified sequence (Figure 1G). Interestingly, 2PyG caused
TeloG23 to fold into an “antiparallel” structure even in K+ solutions
(Figure 1A). This structure, where G23 adopts a syn conformation,19
is normally only observed in Na+-containing solutions. TeloG23
was therefore characterized in Na+ buffer, and it exhibited nearly
the same global structure and thermal stability as the wild-type
sequence in Na+ (∆Tm ) -2 °C, Figure S4B Supporting Informa-
tion). Taken together, these data indicate that 2PyG can be
incorporated into the G-tetrads of these natively folded G-
quadruplexes with little, if any, negative impact on their structure
or stability.20
To study the impact of G-quadruplex folding on the fluorescence
properties of 2PyG, DNA samples were folded in the presence of
variable cation salts. In the presence of K+, Na+, Rb+, or NH4
,
+
three diverse and well-defined G-quadruplex structures were
observed (Figures 1A-B and S4). Surprisingly, the quantum yields
for selective excitation of 2PyG (λex ) 330 nm) in these structures
were 2- to 7-fold higher than that of the 2PyG nucleoside monomer
(2) under the same conditions (Φ ) 0.02, Figures 1G and S1).
The modest quantum yields for 2PyG in ion-folded G-quadruplexes
(Φ ) 0.04-0.15) were compensated by the relatively large molar
extinction coefficient of 2PyG (ꢀ310 nm ≈ 25 000 cm-1 M-1) and its
ability to act as an emissive energy acceptor when base-stacked
with other guanine residues.20 In contrast, the most commonly used
internal probe, 2-aminopurine, is a poor guanine mimic and not
Figure 1. Molar ellipticity (θ) of Telo(wt), TeloG9, and TeloG23 (A) or
cKit(wt) and cKitG10 (B) in 110 mM KCl. Excitation (C) and emission (D)
spectra of TeloG9 and the 2PyG nucleoside in 110 mM KCl or 110 mM LiCl.
Molar ellipticity (E) and excitation spectra (F) of TeloG9 folded in the presence
of 40% PEG, 110 mM NaCl, or 40% PEG and 110 mM NaCl. DNA sequences,
names, quantum yields (Φ), energy transfer efficiencies (ηt), and thermal
denaturation melting temperatures (Tm) of the oligonucleotides studied (G). All
samples contained 2 µM of DNA or 2PyG (2) in an aqueous 10 mM cacodylate
buffer (pH 7.4). Excitation spectra were collected using emission at 415 nm,
and emission spectra were collected using excitation at 260 nm. Thermal melting
temperatures (Tm) were determined by monitoring ∆θ vs temperature. Φ values
and energy transfer efficiencies (ηt) were measured using λex ) 330 or 260
nm, respectively.11,13
very bright due to its low molar extinction coefficient (ꢀ305
)
nm
5600 cm-1 M-1) and low quantum yield (Φ ≈ 0.005) when base-
stacked with guanine residues.7a
The excitation spectra of 2PyG-containing quadruplexes exhibited
maxima at ∼260 and ∼330 nm (Figures 1C and S1). The excitation
peak at 260 nm revealed the presence of DNA-to-2PyG energy
transfer.7b,e Interestingly, the efficiencies of energy transfer (ηt) were
always much higher in G-quadruplex-folded structures as compared
to the same, mostly unfolded DNAs prepared in LiCl-containing
buffers (Figure 1C-D,G).11 Due to their small size and large
desolvation energies, lithium ions are not normally compatible with
G-quadruplex folding.1 DNA samples prepared in aqueous 110 mM
LiCl therefore gave CD data typical of G-stacked single-stranded
DNA (Figure S4). Energy transfer efficiencies were 70-350%
higher for cKitG10, TeloG23, and TeloG9 G-quadruplexes when
folded in 110 mM solutions of Na+, K+, NH4+, and Rb+ (ηt )
0.11-0.41) as compared to the same DNAs prepared in 110 mM
Li+ (ηt ) 0.05-0.11, Figure 1G).13,21 The combination of strong
DNA molar absorptivity (ꢀ260 nm ≈ 250 000 cm-1 M-1) with efficient
energy transfer resulted in fluorescence enhancements up to 15-
fold for ion-containing G-quadruplexes as compared to the corre-
sponding unfolded DNAs in LiCl (Figures 1D,G and S1-S2).
To dissect the multiple roles that metal ions might play in
mediating both G-quadruplex folding and energy transfer, we
prepared G-quadruplexes under salt-deficient conditions (10 mM
9
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C O M M U N I C A T I O N S
lithium cacodylate) by molecular crowding/partial dehydration in
40% polyethylene glycol 200 (PEG).22 Under these conditions,
cations are absent or only weakly bound in the central cavities of
each G-quadruplex.22 For all DNAs evaluated, PEG-mediated
folding resulted in G-quadruplex structures exhibiting cooperative
and reversible folding, as well as highly variable thermal stabilities
(Figure 1E,G). Interestingly, very little energy transfer was observed
in the PEG-folded G-quadruplexes under salt-deficient conditions
(ηt ≈ 0.02-0.07, Figure 1F,G). Quadruplexes prepared in the
presence of both PEG and 110 mM Na+, in contrast, exhibited
energy transfer efficiencies similar to that for the samples prepared
in 110 mM Na+ (ηt ≈ 0.30, Figure 1F,G). Nonspecific electrostatic
stabilization of backbone phosphate repulsion is not responsible
for the enhanced energy transfer, because PEG-folded G-quadru-
plexes prepared in 110 mM of tetrabutylammonium chloride
exhibited approximately the same energy transfer efficiencies as
the PEG-only samples. Taken together, these results suggest that
the G-quadruplex structure by itself is not sufficient for promoting
efficient energy transfer and that ions coordinated directly to the
O6 position of guanine residues play a critical role in mediating
energy transfer reactions in G-quadruplex structures.
electronically resemble emissive enol forms of guanine more than
the corresponding ion-free guanine residues.
Acknowledgment. We thank Professor Andrea Vasella, Dr.
Fabio De Giacomo, and Dr. Thomas Steinlin for providing
8-substituted guanines for initial fluorescence studies.25 Funding
was provided by the University of Zu¨rich Forschungskredit
#57131901 and the Swiss National Science Foundation #130074.
Supporting Information Available: The synthesis and characteriza-
tion of 2-3, additional fluorescence and CD data, and the procedures
for all experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Previous studies proposed that G-G base-stacking, hydrogen
bonding, and/or restricted motions within G-quadruplex structures
can enhance the photoexcited lifetimes and quantum yields of
guanine residues.6 To evaluate the impact of cation binding on the
quantum yields of unmodified guanine residues, we prepared
unmodified G-quadruplexes from Telo(wt) in the presence of 40%
PEG under salt-deficient conditions or in the presence of 40% PEG
and 110 mM NaCl. While both samples were more fluorescent than
a comparable mixture of nucleotide monophosphates,11 samples
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for guanine residues in G-quadruplex structures.6 The enhanced
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the unusually efficient DNA-to-2PyG energy transfer reactions
reported here.21
In summary, the incorporation of a single pyridine unit into
G-quadruplexes has provided a useful fluorescent probe for
monitoring G-quadruplex folding and revealed efficient intramo-
lecular energy transfer reactions in ion-containing G-quadruplex
structures. We used 2PyG in conjunction with PEG-promoted
G-quadruplex folding to dissect the multiple roles metal ions play
in mediating both G-quadruplex folding and energy transfer. Given
the growing interest in G-quadruplex-based devices and materials,3,5
these results may provide important design principles toward
harnessing the potentially useful photophysical properties of G-
quadruplex wires and related structures.5,6 Elucidating the exact
mechanism of cation-mediated energy transfer will require ad-
ditional studies, but our results suggest that the enhanced quantum
yields of unmodified guanine residues upon O6 ion coordination
are responsible for the unusually efficient energy transfer reactions
mediated by G-quadruplex structures.21 Interestingly, the O6
carbonyl stretching frequencies of guanine residues are known to
decrease upon NH4+-mediated quadruplex folding,23 and the
“trapped enol” mimic O6,9-dimethylguanine is known to be highly
fluorescent when protonated.24 Taken together, these results suggest
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(11) See Supporting Information for synthetic and experimental details.
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